Expression of arginase I in myeloid cells limits control of residual disease after radiation therapy of tumors in mice

An accumulating body of evidence demonstrates that radiation therapy can generate adaptive immune responses that contribute to tumor control. However, in the absence of additional immune therapy, the adaptive immune response is insufficient to prevent tumor recurrence or affect distant disease. It has been shown in multiple models that tumor-infiltrating myeloid cells exhibit alternative activation phenotypes and are able to suppress adaptive immune responses, and recent data suggests that the myeloid response in tumors treated with cytotoxic therapy limits tumor control. We hypothesized that tumor myeloid cells inhibit the adaptive immune response after radiation therapy through expression of the enzyme arginase I. Using a myeloid cell-specific deletion of arginase I in mice, we demonstrate an improved tumor control after radiation therapy. However, tumors still recurred despite the conditional knockdown of arginase I. Since multiple alternative factors may combine to inhibit adaptive immunity, we propose that targeting macrophage differentiation may be a more effective strategy than targeting individual suppressive pathways

Expression of NF-κB p50 in Tumor Stroma Limits the Control of Tumors by Radiation Therapy

Radiation therapy aims to kill cancer cells with a minimum of normal tissue toxicity. Dying cancer cells have been proposed to be a source of tumor antigens and may release endogenous immune adjuvants into the tumor environment. For these reasons, radiation therapy may be an effective modality to initiate new anti-tumor adaptive immune responses that can target residual disease and distant metastases. However, tumors engender an environment dominated by M2 differentiated tumor macrophages that support tumor invasion, metastases and escape from immune control. In this study, we demonstrate that following radiation therapy of tumors in mice, there is an influx of tumor macrophages that ultimately polarize towards immune suppression. We demonstrate using in vitro models that this polarization is mediated by transcriptional regulation by NFκB p50, and that in mice lacking NFκB p50, radiation therapy is more effective. We propose that despite the opportunity for increased antigen-specific adaptive immune responses, the intrinsic processes of repair following radiation therapy may limit the ability to control residual disease

Radiation therapy of tumors.

<p>a) C57BL/6 mice were challenged with 2×10⁵ Panc02 <i>s.c.</i> in the right leg and mice received 3 daily doses of 20 Gy focal radiation to the leg beginning on day 14 (RT) or were left untreated (NT). i-ii) 1 day or iii-iv) 7 days following the final radiation dose, tumors were harvested, digested and clonogenic assays performed. b) One and seven days following the final radiation dose, tumors were harvested and tumor-infiltrating cells determined by FACS analysis. Graphs show the mean and standard error of tumor infiltrating CD11b⁺ cells in Panc02 tumors, and include data from two replicate experiments. c) Panc02 tumors were harvested for histology 7 days following the final radiation dose. Images show representative regions of neighboring sections from tumors receiving NT (i & iii) or RT (ii & iv) that were i-ii) H&E stained or iii-iv) underwent immunofluorescence staining with antibodies specific for VWF and F4/80, and detected with antibodies conjugated to AF488 (Green) and AF568 (Red), respectively. Nuclear material was counterstained with DAPI (Blue) and sections were imaged by confocal microscopy.</p

Cytokine responses of tumor macrophages.

<p>a) CD11b⁺IA⁺ cells were sorted from i) 4T1 tumors or ii) from Panc02 tumors and treated <i>in vitro</i> with 100 ng/ml LPS, or left untreated (NT). 24 hours later supernatants were collected and ELISA tested for secretion of IL-10 and TNFα. b) i) Bone marrow-derived macrophages were incubated alone or with untreated or irradiated 4T1 cancer cells for 24 hours before stimulation with 100 ng/ml LPS. Supernatants were collected and ELISA tested for secretion of IL-10 and TNFα after a further 48 hours. ii) Raw264.7 macrophages were incubated alone or with an equal number of untreated or irradiated 4T1 cancer cells (10 or 20 Gy) for 24 hours, then treated with 100 ng/ml LPS and supernatants collected and ELISA tested for secretion of IL-10 after a further 48 hours. Graphs are representative of multiple replicate experiments.</p

Gene expression microarray of tumor macrophages following radiation.

<p>a) Panc02 tumors were harvested 1 and 7 days following the final radiation dose and i) gated CD11b⁺ cells were FACS sorted according to expression of ii) Gr1 and IA. Sorted populations of CD11b⁺Gr1^hi and CD11b⁺IA⁺ cells from iii) NT or iv) RT tumors were tested for morphology by cytopsin and Diff-Quik staining. RNA was prepared from sorted CD11b⁺IA⁺ cells and Affymetrix gene expression microarray analysis was performed. Gene expression profiles were analyzed for the expression of b) lineage markers and c) M1 and M2-associated markers. d) CD11b⁺Gr1^hi and CD11b⁺Gr1^lo cells were sorted as in a) and lysates prepared from sorted cells for western blotting. The image represents 3 western blots cropped and positioned above each other to show detection of Arginase I, iNos and GAPDH. Gene array analysis uses RNA collected from purified macrophages isolated in 2 replicate experiments. Each gene list is sorted by gene expression level and includes an individual key showing the gene intensity scale for that group.</p

Radiation therapy of tumors in NFκB1 knockout mice.

<p>a) Wild-type (wt) or NFκB1^−/− C57BL/6 mice were challenged with 2×10⁵ Panc02 <i>s.c.</i> in the right leg and mice received 3 daily doses of 20 Gy focal radiation to the leg beginning on day 14 (RT) or were left untreated (NT).Tumor leg diameter was measured 3× per week. b) Survival curves of mice in a replicate experiment treated as in a) where mice were euthanized at a leg diameter exceeding 12 mm. c) Proportion of tumors growing following s.c. injection of 3LL or Panc02 in NFκB1^−/− naïve mice or NFκB1^−/− mice that were cured of their primary tumor with radiation therapy (RT).</p

Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized

Listeria vaccination of mice during myeloid contraction.

<p>Flow cytometry of intracellular IFNγ production in response to a) LLO91 or b) AH1 peptide, from mouse spleens 7 days following Listeria vaccination of i) naïve mice, ii) tumor bearing mice left untreated, iii) tumor bearing mice with their tumor irradiated prior to vaccination, iv) control tumor-bearing mice not receiving vaccination. c) Summary of intracellular IFNγ production in response to each peptide, each symbol represents one mouse. Data represents combined data from 3 replicate experiments. d) representative staining showing i) dual IFNγ-CD40L, ii) dual IFNγ-TNFα or iii) triple IFNγ-CD40L-TNFα positive cells from vaccinated mice in response to LLO91 peptide. Summary of single, dual and triple positive IFNγ⁺ cells from each vaccinated group stimulated with iv) LLO91 and v) AH1 peptides. NS = Not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001.</p